Nucleic acid sequence data is valuable in myriad applications in biological research and molecular medicine, including determining the hereditary factors in disease, in developing new methods to detect disease and guide therapy (van de Vijver et al. (2002) “A gene-expression signature as a predictor of survival in breast cancer,” New England Journal of Medicine 347: 1999-2009), and in providing a rational basis for personalized medicine. Obtaining and verifying sequence data for use in such analyses has made it necessary for sequencing technologies to undergo advancements to expand throughput, lower reagent and labor costs, and improve accuracy (See, e.g., Chan, et al. (2005) “Advances in Sequencing Technology” (Review) Mutation Research 573: 13-40, and Levene et al. (2003) “Zero Mode Waveguides for Single Molecule Analysis at High Concentrations,” Science 299: 682-686), the disclosures of which are incorporated herein in their entireties for all purposes.
Single molecule real-time sequencing (SMRT) is a highly parallel sequencing-by-synthesis technology that permits the simultaneous surveillance of, e.g., thousands of sequencing reactions in arrays of multiplexed detection volumes, e.g., zero-mode waveguides (ZMWs). (See e.g., Levene et al. (2003) Zero-mode waveguides for single-molecule analysis at high concentrations, Science 299:682-686; Eid, et al. (2009) Real-Time DNA Sequencing from Single Polymerase Molecules, Science 323:133-138; Published U.S. Patent Application No. 2003/0044781; and U.S. Pat. No. 6,917,726, the disclosures of which are incorporated herein in their entireties for all purposes). Each detection volume in an array creates an illuminated visualization chamber that is small enough to observe the template-dependent synthesis of a single single-stranded DNA molecule by a single DNA polymerase.
When a particular base in the template strand is encountered by the polymerase during the polymerization reaction, e.g., in a ZMW, the enzyme complexes with an available fluorescently labeled nucleotide or nucleotide analog and incorporates that nucleotide or nucleotide analog into the nascent growing nucleic acid strand. During this time, the fluorophore emits fluorescent light whose color corresponds to the nucleotide's or analog's base identity. The polymerase cleaves the bond linking the fluorophore to the nucleotide or analog during the nucleotide incorporation cycle, permitting the dye to diffuse out of the detection volume. The signal returns to baseline, and the process repeats.
A single molecule sequencing reaction is typically localized to a detection volume by immobilizing a DNA polymerase enzyme within or proximal to the site at which the reaction takes place. Ideally, the immobilized polymerase retains its activity and can be used repeatedly and continuously in multiple sequencing reactions. However, it has been observed that in some cases, the processivity, accuracy, and/or activity of the polymerase enzyme can decrease. In particular, in at least some cases, damage to the DNA polymerase, e.g., by exposure to optical energy during fluorescent or chemiluminescent detection, can have a detrimental effect on the enzyme's activity.
Current strategies for single molecule sequencing-by-synthesis employ a polymerase that has been tethered within or proximal to a reaction region within a detection volume, e.g., in a ZMW. What is needed in the art are new methods and compositions that can maintain the processivity, accuracy, and polymerase activity in, e.g., a single-molecule sequencing reaction, while still localizing the polymerization reaction to a defined observation volume. The invention described herein fulfills these and other needs, as will be apparent upon review of the following.